Our long-term goal is to understand the detailed mechanisms of the functional role of K channels in the inner ear. The importance of K channels is underpinned by the fact that mutations of KCNQ channels results in deafness in humans as seen in Jervell Lang Nielson syndrome (JLNS), and an autosomal dominant form of nonsyndromic progressive hearing loss (PHL: DFNA2). To understand the underlying mechanisms of KCNQ channel functions, we are conducting experiments that are revealing surprising, yet insightful results that may delineate the cellular and molecular mechanisms of the channel in normal and in disease states. A diverse class of functionally distinct form of alternatively-spliced KCNQ4 channels is expressed in the cochlea with distinct tonotopic profiles. However, because the channels' form heteromultimers, a dominant-negative form of one KCNQ channel cripples the function of the entire class of K channels in the inner ear. Even more surprising is the finding that different splice variants of the channel have a paradoxical mechanism for K+ extrusion; some of the KCNQ4 isoforms are more efficient in K+ extrusion when the external K+ concentration increases, making the channel a key protein in K+ regulation in the inner ear. In Aim 1, we hypothesize although KCNQ2-5 channels are derived from different genes, the channels interact to produce diverse current phenotypes in the inner ear to confer a pivotal role a K+ homeostasis. Because of the unique interaction, a dominant negative (DN) version of one form cripples the entire class of KCNQ2-5 in the inner ear. We will clone the inner ear-specific channels, localize their differential expression and determine the molecular determinants of their functions. The mechanisms of modulation of KCNQ4 by interacting protein partners and second messengers will also be assessed. In Aim 2, we hypothesize that differential expression of different isoforms/alternative splice variants is the underlying mechanisms for the base-to-apex PHL seen in mutations of KCNQ4 channels. Finally, we will determine the functional determinants of the channel that allow it to extrude K+ in increased external K+. Thus, testing the hypothesis that crucial to the role of KCNQ4 channels in the inner ear is their paradoxical feature with some isoforms having enhanced extrusion of K+ even in the presence of increased extracellular K+. We will use molecular biological, (cloning) biochemical (yeast 2-hybrid systems and siRNA), and functional electrophysiological techniques to address the Aims of the proposal. All experiments will use mice as model. Collectively, these studies will substantially expand our understanding of the cellular mechanisms for the regulation of K+ in the inner ear. Understanding the role of KCNQ channels and their associated proteins in the inner ear is necessary to design therapies for hearing loss (e.g. PHL).7 7. Project Narrative Our study will directly examine the molecular mechanisms of the functional coupling of KCNQ channels with other binding proteins and examine differential isoform-specific subcellular localization of the channels in mice cochlea HCs and SGNs. We will also identify the novel interacting proteins and their functional significance. The study will have important implications in our understanding of how point mutations of a single animo acid (aa) in the channel can cripple an entire family of KCNQ channels and lead to PHL. Indeed, the findings may transcend inner ear-specific KCNQ channel functions. Because the channel produces characteristic muscarinic (M)-type currents in several sensory neurons, these studies will address fundamental uncertainties that surround the functions of the channels throughout the nervous system.